Monoclonal antibodies have been proven to be effective reagents for both diagnosis, prevention and therapy of disease (Glennie and Johnson, Immunol. Today, 21, p. 403-410, 2000). This is due to their unique capacity to bind very specifically to particular epitopes on target molecules (antigens) via their variable domains and, at the same time, to mediate effector functions via their constant region domains (Frazer and Capra, Fundamental Immunology, W. E. Paul (Ed.), Fourth Edition, p. 37-74, 1999) (FIG. 1). This enables monoclonal antibodies to specifically identify unique antigens in complex mixtures of macromolecules and to direct effector functions to these targets.
Antibodies (or immunoglobulins) consist of two identical heavy (H) chain and light (L) chain glycoproteins that are linked via disulphide bonds (FIG. 1). Each H and L chain comprises an N-terminal variable domain that varies between different antibodies and a C-terminal constant region, that is identical in different antibodies belonging to the same immunoglobulin isotype (FIG. 1). The combination of H and L chain variable domains generates the antigen binding pocket of the antibody and determines its specificity (or idiotype), whereas the constant regions determine its isotype (Frazer and Capra, s.a.). The variability of immunoglobulins results from the fact that VH and VL domains are encoded by a multitude of gene segments, that are designated V (variable), D (diversity; only present in the H chain locus), and J (joining) gene segments (Tonegawa, Nature, 302, p. 575-581, 1983) (FIG. 1). During the differentiation of B lymphocytes one V, D and J gene segment is randomly selected in each cell for the site-specific recombination process of V(D)J recombination, that assembles the gene segments, such that new coding regions for VH or VL domains are generated (Grawunder et al., Curr. Opin. Immunol., 10, p. 172-180, 1998). Due to the multitude of V, D, and J gene segments, and imprecision in gene segment joining, an enormous repertoire of different V region specificities can be generated by the millions of B lymphocytes produced by the immune system every day (Melchers et al., Curr. Opin. Immunol., 7, p. 214-227, 1995).
During evolution, immunoglobulin genes have slightly diverged between different species, such that the constant regions of antibodies differ between species. As a consequence, immunoglobulins from one species are most often immunogenic, if introduced into the vascular system of another species.
The immunogenicity of xenogeneic monoclonal antibodies therefore limits their use in the therapy of human disease, because exposure of patients to xenogeneic antibodies may result in adverse effects, even acute toxicity, or might simply lead to the neutralization and clearance of the applied antibody, thereby reducing its pharmacological efficacy (Clark, Immunol Today, 21, p. 397-402, 2000) In contrast, administration of fully human antibodies to patients usually does not lead to any of the aforementioned complications.
Human antisera or human polyclonal antibodies have occasionally been isolated from the blood of individual patients for the treatment or prevention of rare and usually highly lethal diseases, like Ebola virus infections, or e.g. for the treatment of individuals after exposure to snake venoms. However, this approach, for many reasons, is impractical for the treatment of diseases affecting larger populations. Furthermore, for ethical reasons, it is impossible to immunize humans with a given antigen for the purpose of monoclonal antibody production, because the B-lineage cells in humans producing the desired antibodies develop and reside in secondary lymphoid organs and cannot easily be obtained. Furthermore, many potential target antigens for therapeutic antibodies, in particular for cancer therapy, are human proteins (Glennie and Johnson, s.a.). Under normal circumstances, humans will not develop antibodies against these targets. Therefore, substantial efforts have been made in the recent past to develop procedures for the development of therapeutic human or humanized antibodies without the need of the human immune system.
The simplest approach uses standard genetic engineering techniques for cloning of cDNAs encoding the variable domains of H and L chains (VH and VL domains) from a hybridoma secreting a xenogeneic antibody of desired specificity. The cloned variable region cDNAs are then cloned into appropriate E. coli, yeast, insect or mammalian cell expression vectors containing human constant region genes. This will allow the production of monoclonal antibodies with the xenogeneic VH and VL domains fused to the human CH and CL constant region domains, thereby resulting in a humanized antibody. However, this approach has the disadvantage that the fusion of xenogeneic VH and VL domains to human CH and CL constant regions may result in either decreased affinity or altered specificity. In addition, the heterologous VH and VL of the humanized antibody are still immunogenic in humans, because framework regions of V domains vary between different species. This problem can in some cases be circumvented by grafting individual complementarity determining regions (CDRs), which mediate the direct contact to antigens, onto human framework regions of H and L chain variable domains (Fiorentini et al., Immunotechnology, 3, p. 45-59, 1997). However, for unknown reasons, CDR grafting still often results in the generation of immunogenetic antibodies and/or the decrease/loss of affinity and specificity.
Therefore, two different and more effective approaches have been developed in recent years for the generation of completely human immunoglobulins.
The first method is based on the expression (display) of single chain variable regions (scFv) consisting of one VH and VL domain on the surface of filamentous bacteriophages of E. coli (Hoogenboom and Chames, Immunol. Today, 21, p. 371-3818, 2000) (cf. also EP 0 585 287 B1, EP 0 605 522 B1). Phage display libraries containing a diverse repertoire of VH and VL chain can be constructed, and individual scFv specificities can be isolated from such libraries by binding of recombinant phages to immobilized antigen (McCafferty et al., Nature, 348, p. 552-554, 1990).
Phage display libraries for scFv binding proteins derived from a natural human repertoire have the drawback of being restricted to specificities contained in that repertoire, and specificities for many human antigens are therefore most often not represented in these libraries. Although combinatorial or synthetic phage display libraries may be used to circumvent this problem, a general drawback of this technology is that high affinity binding proteins for a given antigen can often not be isolated. Therefore, substantial efforts have been invested in constructing very large primary libraries by either brute force cloning or site specific recombination procedures. The The best combinatorial or synthetic libraries are estimated to contain 109-1011 potentially different binding sites and can yield binding specificities in the 1-200 nM range (Hoogenboom and Chames, s.a.). However, higher affinities in the picomolar range (10−12 M), that can be reached during affinity maturation of immunoglobulins in germinal center B cells in vivo, can only be generated employing additional tedious genetic engineering procedures, including V gene shuffling, error-prone PCR, the use of E. coli mutator strains etc.
Whatever the outcome of a phage display library selection process is, eventually the genes encoding the scFv binding site need to be recloned into suitable expression vectors for human immunoglobulins, and these need to be stably transferred into a cellular expression system allowing the large-scale production of human antibodies, which is another time-consuming and expensive procedure.
The second approach for the production of fully human monoclonal antibodies is based on the use of transgenic mice harbouring constructs for the human immunoglobulin H and L chain gene loci (Jakobovits, Curr. Opin. Biotechnol., 6, p. 561-566, 1995). The human immunoglobulin transgenes are eventually bred onto a genetic background that does no longer allow assembly of the endogenous murine antigen receptor genes. In these mice, development of B-lineage cells depends on the expression of immunoglobulin H and L chains from the human transgenic constructs, and B lineage cells of these mice are only capable of producing human antibodies. There are three variations of this technique. In one, human immunoglobulin miniloci are used as transgenes, from which only a limited repertoire of human antibodies can be generated (Fishwild et al., Nat. Biotechnol., 14, p. 845-851, 1996) (also cf. to, EP 0 546 073 B1, U.S. Pat. No. 5,874,299, WO 92/03918). In a second variation, large regions of the human IgH and IgκL chain gene loci encompassing the majority of the variable region gene segments cloned into yeast artificial chromosomes are used. In a third variation, pieces of human chromosomes containing the entire human immunoglobulin H and L chain gene loci are integrated into the germline of mice generating so-called trans-chromosomic animals. Mice with such complex transgenes develop a practically normal human immunoglobulin repertoire.
Immunization of these transgenic or trans-chromosomic mice results in a humoral immune response resulting in the generation of fully human antibodies. This approach of generating human antibodies has one important advantage over the phage display library technology: High affinity antibodies for a given antigen can be generated, because the human immunoglobulins can undergo affinity maturation in germinal center B cells, which is a normal process in the course of an immune reaction.
However, there is one important drawback of this technology: First, the generation of transgenic or trans-chromosomic mice is very tedious, difficult, time-consuming, and therefore relatively expensive. For example, WO 92/03918 discloses B cells of a transgenic mouse expressing human monoclonal antibodies. In order to achieve this transgenic mouse, fertilized eggs are transfected with exogenous elements by introducing the heterologous IgH and IgL chain transgenes by pronuclear injection. The entire procedure requires the generation of at least four different mouse strains: two strains with targeted disruptions within the endogenous IgH and IgL chain gene loci, as well as two strains carrying transgenes or trans-chromosomes for part or all of the human IgH and IgL chain gene loci. In addition, all these four mouse strains have to be bred together, resulting in mice with a genotype carrying homozygous disruptions of both the endogenous IgH and IgL chain gene loci and at the same time carrying the human transgenes encoding both the heterologous IgH and IgL chains. Generation of the different knock-out and transgenic mouse strains, their screening and eventual crossbreeding is a lengthy procedure that will require at least two years, even if each step is completely optimized. The extensive time-frames required for the development of a xenomouse strain therefore leaves little flexibility for designing different mouse strains containing modified transgenic constructs. Therefore, this technology is not suitable for the modification and improvement of existing antibodies (e.g. of their affinity), because this would require the lengthy procedure of generating a novel transgenic mouse strain for this one antibody. This technology is therefore basically restricted to the de novo generation of human antibodies.
Based on the aforementioned facts, there is clearly the need for a technology allowing the production of fully human antibodies that would combine the advantages of both the phage display system (i.e. speed and flexibility in generating human antibodies, and the ability to modify and improve the properties of existing antibodies), and of the human immunoglobulin transgenic mouse technology (i.e. the ability to obtain high affinity antibodies due to affinity maturation occurring in the immune system, and production of antibodies with physiologic and natural structural features).